www.elsevier.com/locate/foodchem

Food Chemistry 92 (2005) 119–124

FoodChemistry

Antioxidant and antibacterial activities of variousseabuckthorn (Hippophae rhamnoides L.) seed extracts

P.S. Negi a, A.S. Chauhan b,*, G.A. Sadia b, Y.S. Rohinishree b, R.S. Ramteke b

a Human Resource Development Department, Central Food Technological Research Institute, Mysore 570 020, Indiab Fruit and Vegetable Technology Department, Central Food Technological Research Institute, Mysore 570 020, India

Received 2 October 2003; received in revised form 16 July 2004; accepted 16 July 2004

Abstract

Seabuckthorn (Hippophae rhamnoides L.) seeds were successively extracted with chloroform, ethyl acetate, acetone and methanol

(MeOH) using a Soxhlet extractor for 8 h each. The crude extracts were screened for antioxidant and antibacterial activities. The

reducing power and antioxidant activities evaluated in various in vitro models (1,1-diphenyl-2-picrylhydrazine and liposome model

system) showed the highest activity for MeOH extract. The MeOH extract was also found to posses maximum antibacterial activity.

The MIC values, with respect to MeOH extract for Bacillus cereus, Bacillus coagulans, Bacillus subtilis, Listeria monocytogenes, Yer-

sinia enterocolitica, were found to be 200, 300, 300, 300, and 350 ppm, respectively. These results indicated the possibility of using

seabuckthorn seeds for medicinal uses and food preservation.

� 2004 Elsevier Ltd. All rights reserved.

Keywords: Hippophae rhamnoides; Antioxidant activity; Antibacterial activity; Seed extract; Methanolic extract

1. Introduction

Seabuckthorn (Hippophae rhamnoides L., Elaeagna-

ceae) is a native of Eurasia and has been domesticated

in several countries including India, China, Nepal, Paki-

stan, Myanmar, Russia, Britain, Germany, Finland,

Romania and France at a high altitude of 2500–4300

m. It is known as the wonder plant and bears small or-ange yellow- to red-coloured fruits on two-year-old

thorny twigs. The berry-like fruit develops from an

ovary, or calyx tube connected to an ovary. Seabuck-

thorn (SBT) berries are known to be acidic with a mild

sweet unique aroma. High amounts of vitamin C, flavo-

noids, oils and oil-soluble compounds, as well as miner-

als, are present in the berry (Kallio, Yang, Tahvonen, &

Hakala, 2000). Berries also contain many bioactive sub-

0308-8146/$ - see front matter � 2004 Elsevier Ltd. All rights reserved.

doi:10.1016/j.foodchem.2004.07.009

* Corresponding author. Tel.: +91 0821 2515653; fax: +91 0821

2517233.

E-mail address: attar2k@hotmail.com (A.S. Chauhan).

stances and can be used in the treatment of several dis-

eases, such as cardiovascular disease, cancer, and acute

mountain sickness. For the past 50 years several medic-

inal preparations of SBT have been clinically used to

treat radiation damage, burns, oral inflammation and

gastric ulcers in China and the former Soviet republic,

and more than 300 preparations have been reported in

literature (Lu, 1992). In addition to medicinal use, theberries are processed into various products such as juice

and marmalade, and used for flavouring of dairy prod-

ucts because of their unique taste (Gao, Ohlander,

Jeppsson, Bjork, & Trajkovski, 2000).

Spoilage of foods due to the presence of bacterial and

fungal infection has been a major concern for decades

and it causes a considerable loss worldwide. The de-

mand for non-toxic, natural preservatives has been ris-ing with increased awareness and reports of ill-effects

of synthetic chemicals present in foods. Furthermore,

emergence of food-borne pathogens has lately become

a major public health concern. Many compounds

120 P.S. Negi et al. / Food Chemistry 92 (2005) 119–124

present in plants have been reported to be biologically

active, antimicrobial, allopathic antioxidants and have

bioregulatory properties. SBT oils, juice, leaves and

bark are well known for their medicinal properties,

and they have been used to treat high blood lipid symp-

toms, gingivitis, eye and skin ailments, and cardiovascu-lar diseases (Liu, Wu, & Liu, 1980; Yang et al., 2000).

Proximate composition of SBT has been well docu-

mented (Gao et al., 2000). Also, there have been reports

on chemical composition, characterization and health

benefits of SBT fruit and its oil (Chen et al., 1990; Yang

et al., 2000). But, SBT seed, a byproduct of the berry

processing industry, is not yet exploited. To our knowl-

edge, no information is available on the antioxidant andantibacterial properties of SBT seeds. Therefore, the

objective of the present study was to investigate the anti-

oxidant activity of various SBT seed extracts in in vitro

models, and determine their antibacterial activity

against different food-borne pathogens for their poten-

tial as a natural preservative and for nutraceutical

formulations.

2. Materials and methods

2.1. Materials

SBT berries were collected from the Lahaul Spiti re-

gion (Himachal Pradesh, India) and seeds were obtained

after deseeding the berries by passing through stainlesssteel sievers (Mesh pore size 0.042 in.). 1,1-Diphenyl-2-

picrylhydrazyl (DPPH), and tannic acid were obtained

from Sigma Chemical Co. (St. Louis, MO, USA). All

solvents/chemicals used were of analytical grade and ob-

tained from Merck, Mumbai, India.

2.2. Preparation of the SBT seed extracts

Dried seeds of Hippophae rhamnoides were powdered

and successively extracted in a Soxhlet extractor for 8 h

each with chloroform, ethyl acetate, acetone, and meth-

anol according to the procedure of Chauhan, Negi, and

Ramteke (2003). The extracts were used as such for

determination of reducing power, dissolved in metha-

nol: water (6:4 v/v) for evaluation of antioxidant capac-

ity by the liposome model system, in dimethylsulphoxide for scavenging activity by DPPH method;

and in propylene glycol for evaluation of antibacterial

activity.

2.3. Determination of total phenolics

The concentration of phenolics in the extracts was

determined by the method of Negi, Jayaprakasha, andJena (2003) and results were expressed as (+) tannic acid

equivalents. Five milligrammes of each dried SBT seed

extract were mixed with 10 ml mixture of acetone and

water (6:4 v/v). An aliquot of the samples was mixed

with 1.0 ml of 10-fold diluted Folin–Ciocalteu reagent

and 2 ml of 10% sodium carbonate solution. After

standing for 30 min at room temperature, the absorb-

ance was measured at 765 nm using a GBC UV–Vis Spectrophotometer (Model Cintra-10, Victoria,

Australia).

2.4. Evaluation of antioxidant activity by liposome model

systems

Egg lecithin (300 mg) was sonicated with 30 ml phos-

phate buffer (10 mM, pH 7.4) in an ultrasonic sonicatorfor 10 min to ensure proper liposome formation. The

various dried extracts (25–100 lg) of each sample were

mixed with the sonicated solution (0.5 ml, 10 mg/ml),

FeCl3(0.5 ml, 400 mM), and ascorbic acid (0.5 ml, 400

mM). The antioxidative action was measured by the

method of Buege and Aust (1978). The absorbance of

the samples was determined at 535 nm after incubation

for 1 h at 37 �C. The results were expressed as nmol ofmalondialdehyde (MDA) formed per mg lipid and were

calculated by using an extinction coefficient of 1.56 · 105

M�1 cm�1.

2.5. Determination of reducing power

The determination of reducing power was performed

as described by Yen and Duh (1993). Various extracts(0.48, 1.2, 2.4, 3.6, and 4.8 mg) were mixed with phos-

phate buffer (5.0 ml, 2.0 M, pH 6.6) and 1% potassium

ferricyanide (5 ml), and incubated at 50 �C for 20 min;

5 ml of 10% trichloroacetic acid were added, and the

mixture was centrifuged at 2500 rpm for 10 min. The

upper layer of the solution (5 ml) was mixed with dis-

tilled water (5 ml) and 0.1% ferric chloride (1 ml) and

the absorbance was read at 700 nm. Increase in theabsorbance of the reaction mixture indicated increase

in the reducing power.

2.6. Scavenging of 1,1-diphenyl-2-picrylhydrazyl (DPPH)

radical

Free radical-scavenging activity of each antioxidant

was assayed using a stable free radical, DPPH, accord-ing to the method of Blois (1958). The reaction mixture

contained 0.5 ml of 0.5 mM DPPH and 0.1 ml of di-

methyl sulphoxide containing the antioxidant extract

at different concentrations (10–50 lg). Finally, the total

volume of the reaction mixture was adjusted to 1.0 ml by

adding 100 mM Tris–HCl buffer (pH 7.4). The reaction

mixture was allowed to stand for 20 min at room tem-

perature in the dark and the radical-scavenging activityof each antioxidant was quantified by decolorization at

517 nm.

0

10

20

30

40

50

60

Chloroform Ethylacetat Acetone Methanol

Yield (g/100g) Total phenolics (%)

a

a

b

b

b

c

c

d

Fig. 1. Yield and total phenolics in SBT seed extracts: –¤– Yield (g/

100 g), –n– Total phenolics (%). Mean values ± SD followed by same

letter are not significantly different (P < 0.05).

P.S. Negi et al. / Food Chemistry 92 (2005) 119–124 121

2.7. Antibacterial test by pour plate method

Bacterial cultures, namely, Bacillus cereus, B. coagu-

lans, B. subtilis, Listeria monocytogenes and Yersinia

enterocolitica, were obtained from the Department of

Food Microbiology of this Institute. Bacillus cereus, B.coagulans and B. subtilis were grown in nutrient agar

(HiMedia, Mumbai, India), and L. monocytogenes and

Y. enterocolitica were grown in Brain heart infusion agar

(HiMedia, Mumbai, India) at 37 �C. Each bacterial

strain was transferred from stored slants at 4–5 �C to

10 ml broth and cultivated overnight at 37 �C. A precul-

ture was prepared by transferring 1 ml of this culture to

9 ml broth and cultivated for 48 h. The cells were har-vested by centrifugation (1200g, 5 min), washed and sus-

pended in saline.

The SBT seed extracts were tested against different

bacteria by the method of Negi, Jayaprakasha, Rao,

and Sakariah (1999). To flasks containing 20 ml melted

cool agar, different concentrations of test material in

propylene glycol were added. Equivalent amounts of

propylene glycol were used as controls. One hundredll (about 103 cfu/ml) of each bacterium to be tested were

inoculated into the flasks under aseptic conditions. The

media were then poured into sterilized Petri plates, in

duplicate, and incubated at 37 �C for 20–24 h. The col-

onies, developed after incubation, were counted and ex-

pressed as colony forming units per ml of culture (cfu/

ml). The minimum inhibitory concentration (MIC)

was reported as the lowest concentration of the com-pound capable of inhibiting the complete growth of

the bacterium being tested.

2.8. Statistical analysis

All the experiments were repeated three times and the

data were calculated as means ± SD. The data on the

antioxidant effect of different extracts were analyzed bya two factor experiment in a randomized complete block

design with an experiment involving 4 extracts, 5 con-

centrations and 3 replications for the liposome model

system, 3 extracts, 5 concentrations and 3 replications

for the radical-scavenging activity on DPPH, and 4 ex-

tracts, 6 concentrations and 3 replications for reducing

power studies. One-way ANOVA was used to determine

Table 1

Antioxidant activity of SBT seed extracts by the liposome model system A

Concentration (lg/ml) Chloroform Ethyl acetate

0 3.03 ± 0.17a 3.03 ± 0.17a

25 2.86 ± 0.00b 1.83 ± 0.03f

50 2.57 ± 0.00c 1.51 ± 0.04g

75 2.33 ± 0.04d 0.93 ± 0.04i

100 2.15 ± 0.03e 0.58 ± 0.00k

A Antioxidant activity as nmol MDA/mg lipid.Mean values ± standard

(P < 0.05).

the differences in yields and phenolic contents of differ-

ent extracts. The Duncan�s multiple range test (DMRT)

was used for making comparisons (Gomez & Gomez,

1984).

3. Results and discussion

The SBT seeds were extracted successively with chlo-

roform, ethyl acetate, acetone and methanol using a

Soxhlet extractor for 8 h each. The yields of ethyl ace-

tate, acetone and chloroform extracts were significantly

lower than methanol extracts (Fig. 1). The phenolic con-

tents in different extracts also varied significantly. Thenmethanolic extract had the highest phenolic content, fol-

lowed by ethyl acetate and acetone extract. Variations in

the yields and phenolic contents of various extracts are

attributed to polarities of different compounds present

in the seeds and such differences have been reported in

the literature for other fruit seeds (Jayaprakasha, Singh,

& Sakariah, 2001).

Acetone Methanol

3.03 ± 0.17a 3.03 ± 0.17a

1.48 ± 0.035g 1.29 ± 0.00h

1.14 ± 0.02h 0.98 ± 0.02i

0.76 ± 0.05j 0.68 ± 0.04jk

0.55 ± 0.02k 0.34 ± 0.02l

deviations (n = 3) with the same letter are not significantly different

122 P.S. Negi et al. / Food Chemistry 92 (2005) 119–124

The antioxidative actions of SBT seed extracts in the

liposome system, induced by FeCl3plus ascorbic acid

and determined by the thiobarbituric acid method, are

shown in Table 1. Significant differences (P < 0.05) were

found among control and liposome-containing SBT seed

extracts. The chloroform, ethyl acetate, acetone andmethanol extracts at 100 lg showed 2.15, 0.58, 0.54

and 0.35 nmol MDA/mg lipid formation, respectively.

However, the formation of the MDA for the control

was 3.02 nmol MDA/mg lipid, indicating that the chlo-

Table 2

% Radical-scavenging activity on DPPH by various SBT seed

extractsA

Concentration (lg/ml) Ethyl acetate Acetone Methanol

10 13.5 ± 1.44b 9.51 ± 0.02a 40.4 ± 2.24e

20 26.8 ± 2.18c 14.8 ± 0.73b 71.2 ± 0.80h

30 40.0 ± 1.94e 31.4 ± 1.27d 85.2 ± 2.21i

40 43.6 ± 1.39e 41.3 ± 2.75e 92.2 ± 0.68j

50 50.1 ± 5.50f 59.0 ± 4.14g 93.5 ± 0.32j

A Mean values ± standard deviations (n = 3) with the same letter

are not significantly different (P < 0.05).

Fig. 2. Effects of SBT seed extracts on growth of different bacteria: Bacillu

Yersinia enterocolitica.

roform, ethyl acetate, acetone and methanol extracts

inhibited the peroxidation of lecithin by 29.1%, 80.8%,

82.2% and 88.5%, respectively. A similar trend of varia-

ble antioxidant activity for different solvent extracts of

grape seeds was also observed by Jayaprakasha et al.

(2001).It is well known that free radicals play an important

role in autooxidation of unsaturated lipids in foodstuffs.

Oxidation of muscle cholesterol may be initiated by free

Table 3

Reducing power of various SBT seed extractsA

Amount

(mg)

Chloroform Ethyl acetate Acetone Methanol

0.0 0.03 ± 0.00a 0.03 ± 0.00a 0.03 ± 0.00a 0.03 ± 0.00a

0.48 0.17 ± 0.01b 0.38 ± 0.05d 0.62 ± 0.002e 0.74 ± 0.02g

1.20 0.26 ± 0.00c 0.68 ± 0.01f 1.21 ± 0.00j 1.50 ± 0.00k

2.40 0.58 ± 0.01e 0.98 ± 0.01i 1.87 ± 0.01m 2.14 ± 0.00n

3.60 0.65 ± 0.00f 1.23 ± 0.02j 2.61 ± 0.00o 2.87 ± 0.00p

4.80 0.81 ± 0.00h 1.64 ± 0.01l 3.10 ± 0.00q 3.62 ± 0.00r

A Absorbance at 700 nm. Mean values ± standard deviations (n = 3)

with the same letter are not significantly different (P < 0.05).

s cereus, Bacillus coagulans, Bacillus subtilis, Listeria monocytogenes,

P.S. Negi et al. / Food Chemistry 92 (2005) 119–124 123

radicals generated during the oxidation of polyunsatu-

rated fatty acids (Hoelscher, Savell, Smith, & Cros,

1988). DPPH is used as a free radical to evaluate anti-

oxidative activity of some natural sources, and the

degree of its discoloration is attributed to hydrogen-

donating ability of test compounds, which is indicativeof their scavenging potential (Shimada, Fujikawa, Ya-

hara, & Nakamura, 1992). In the present work, also,

the comparison of scavenging effects of ethyl acetate,

acetone and methanol extracts on DPPH radical showed

the consistently higher radical-scavenging activity for

methanolic extract at all the concentrations (Table 2).

The data show that the extracts are free radical inhibi-

tors and act as primary antioxidants. These resultsconfirm earlier studies, where inhibition of chromium-

induced free radicals was reported by SBT leaf and fruit

methanolic extracts (Geetha, Sai Ram, Singh, Ilavazha-

gan, & Sawhney, 2002).

Table 3 shows the reducing powers of different SBT

seed extracts using the potassium ferricyanide method

at various extract concentrations. It appears that anti-

oxidative activity may have a mutual correlation withthe reducing effect. Chloroform extract showed the least

reducing power and methanol extract had the highest

activity. Jayaprakasha et al. (2001) also noted differ-

ences in reducing power of various extracts of grape

seed. The reducing properties are generally associated

with the presence of reductones and Gordon (1990) re-

ported that the antioxidant activity of reductones is be-

lieved to break radical chains by donation of a hydrogenatom, indicating that the antioxidative properties are

concomitant with the development of the reducing

power. Therefore, the marked antioxidative activity in

MeOH extract may be associated with its higher reduc-

ing power.

The effect of SBT seed extracts on growth of different

bacteria is presented in Fig. 2. Different extracts inhibited

growth to variable extents, depending on the bacteriumin question. Methanolic extract was found to be the most

effective, followed by chloroform and acetone extracts.

Yersinia enterocoliticawas the most resistant to all the ex-

tracts, and higher MIC values were obtained for it. High-

er resistance of Gram-negative bacteria to external agents

has been earlier reported, and it is attributed to the pres-

ence of lipopolysaccharides in their outer membranes,

which make them inherently resistant to antibiotics,detergent and hydrophilic dyes (Nikaido & Vaara,

1985). Similar trend for inhibition of bacterial growth

have been observed in earlier studies with SBT berry ex-

tracts (Puupponen-Pimla et al., 2001) and other plant ex-

tracts (Negi & Jayaprakasha, 2003). The reason for

higher sensitivity of the Gram-positive bacteria than neg-

ative bacteria could be ascribed to the differences between

their cell wall compositions. The Gram-positive bacteriacontain an outer peptidoglycone layer, which is an inef-

fective permeability barrier (Scherrer & Gerhardt, 1971).

4. Conclusions

We propose that the higher antibacterial and antioxi-

dant activities of methanol extract of SBT seeds may be

due to the higher phenolic content. Patro et al. (2001)

also concluded that higher antioxigenic activity of SBTberry is due to higher contents of flavonoids. Recently

Rosch, Bergmann, Knorr, and Kroh (2003) observed

that, although a few phenolic compounds present in

SBT fruit juice have good antioxidant capacity, their

contribution to the antioxidant effect is meagre in com-

parison to ascorbic acid. As our extraction procedure

has eliminated the presence of ascorbic acid, we suppose

that selective extraction and concentration of phenoliccompounds in methanolic extract of SBT seed might

have resulted in higher biological activity. Thus, the re-

sults indicate that selective extraction from natural

sources, by an appropriate solvent, is important for

obtaining fractions with high antioxidant and high anti-

bacterial activity. The antibacterial and antioxidant

properties of SBT seeds can add value to this wonder

plant as, at present, except for the seeds, all parts ofthe plant are being utilized. This is a preliminary report

on the isolation of antioxidants and antibacterial frac-

tions from SBT seed, and further studies are needed

for the characterization of individual phenolic com-

pounds to elucidate the mechanisms underlying bioac-

tive properties and the existence of possible synergism,

if any, among these compounds.

Acknowledgements

We thank Dr. V. Prakash, Director, Dr. K. V. R. Ra-

mana, Head, Fruit and Vegetable Technology and Dr.

M. C. Varadaraj, Head, Human Resource Develop-

ment, Central Food Technological Research Institute,

Mysore, Karnataka, India for constant encouragement.

References

Blois, M. S. (1958). Antioxidant determination by the use of stable free

radicals. Nature, 181, 1199–1200.

Buege, J. A., & Aust, S. D. (1978). Microsomal lipid peroxidation. In

S. Fleischer & L. Packer (Eds.). Methods in enzymology (Vol. L II,

52, pp. 302–310). New York: Academic press.

Chauhan, A. S., Negi, P. S., & Ramteke, R. S. (2003). One step process

for preparing antibacterial and antioxidant fraction from seabucthorn

parts (Hippophae rhamnoides L.). United States Patent Applica-

tion, Ref. No. 0172NF2003/US.

Chen, Y., Jiang, Z., Qin, W., Ni, M., Li, X., & He, Y. (1990). Chemical

composition and characteristics of seabuckthorn fruit and its oil.

Chemistry and industry of forest products (Chinese), 10(3), 163–175.

Gao, X., Ohlander, M., Jeppsson, N., Bjork, L., & Trajkovski, V.

(2000). Changes in antioxidant effects and their relationship to

phytonutrients in fruits of sea buckthorn (Hippophae rhamnoides.

L) during maturation. Journal of Agricultural and Food Chemistry,

48, 1485–1490.

124 P.S. Negi et al. / Food Chemistry 92 (2005) 119–124

Geetha, S., Sai Ram, M., Singh, V., Ilavazhagan, G., & Sawhney, R.

C. (2002). Antioxidant and immunomodulatory properties of

seabuckthorn (Hippophae rhamnoides) – An in vitro study. Journal

of Ethnopharmacology, 79, 373–378.

Gomez, K. A., & Gomez, A. A. (1984). Comparison between treatment

means. InK.A.Gomez&A.A.Gomez (Eds.),Statistical Procedures

for Agricultural Research (pp. 207–240). New York: Wiley.

Gordon, M. F. (1990). The mechanism of antioxidant action in vitro.

In B. J. F. Hudson (Ed.). Food Antioxidants (1, pp. 1–18). London:

Elsevier Applied Science.

Hoelscher, L. M., Savell, J. W., Smith, S. B., & Cros, H. R. (1988).

Subcellular distribution of cholesterol within muscle and adipose

tissue of beef loin steaks. Journal of Food Science, 53, 718–722.

Jayaprakasha, G. K., Singh, R. P., & Sakariah, K. K. (2001).

Antioxidant activity of grape seed (Vitis vinifera) exracts on

peroxidation models in vitro. Food Chemistry, 73, 285–290.

Kallio, H., Yang, B., Tahvonen, R., & Hakala, M. (2000). Compo-

sition of sea buckthorn berries of various origins. In Proceedings

of international workshop on sea buckthorn (pp. 13–19), China,

Beijing.

Liu, B., Wu, Z., & Liu, W. (1980). Preliminary observation on curing

effects of Sea-buckthorn fruit juice for high blood cholesterol and

coronary heart disease. Acta Academiae Medicinae Sichua, 11,

178–182.

Lu, R. (1992). Seabuckthorn: A multipurpose plant species for fragile

mountains. ICIMOD Occasional Paper No. 20 (pp. 62). Khat-

mandou, Nepal.

Negi, P. S., & Jayaprakasha, G. K. (2003). Antioxidant and antibac-

terial activities of Punica granatum peel extracts. Journal of Food

Science, 68, 1473–1477.

Negi, P. S., Jayaprakasha, G. K., & Jena, B. S. (2003). Antioxidant

and antimutagenic activities of pomegranate peel extracts. Food

Chemistry, 80, 393–397.

Negi, P. S., Jayaprakasha, G. K., Rao, L. J. M., & Sakariah, K. K.

(1999). Antibacterial activity of turmeric oil – A byproduct from

curcumin manufacture. Journal of Agricultural and Food Chemistry,

47, 4297–4300.

Nikaido, H., & Vaara, M. (1985). Molecular basis of bacterial outer

membrane permeability. Microbiological Reviews, 49, 1–32.

Patro, B.S., Samuel, R., Sreekanth, A., Dwivedi, S.K., Chaurasia,

O.P., Chattopadhyay, S., et al. (2001). Anti-oxygenic activity of

Indian seabuckthorn (Hippophae rahmnoides L.). In V. Singh, P.K.

Khosla (Eds.), Seabuckthorn – A resource for health and environ-

ment in twenty first century. Proceedings of international workshop

on seabuckthorn (pp. 194–198), Feb. 18–21. CSKHPAU, Palampur

and Indian Society of Tree Scientists, Solan, India.

Puupponen-Pimla, R., Nohynek, L., Meier, C., Kahkonen, M.,

Heinonen, M., Hopia, A., et al. (2001). Antimicrobial properties

of phenolic compounds from berries. Journal of Applied Microbi-

ology, 90, 494–507.

Rosch, D., Bergmann, M., Knorr, D., & Kroh, L. W. (2003).

Structure-antioxidant efficiency relationships of phenolic com-

pounds and their contribution to the antioxidant activity of

seabuckthorn juice. Journal of Agricultural and Food Chemistry,

51, 4233–4239.

Scherrer, R., & Gerhardt, P. (1971). Molecular sieving by the Bacillus

megatrium cell wall and protoplast. Journal of Bacteriology, 107,

718–735.

Shimada, K., Fujikawa, K., Yahara, R., & Nakamura, T. (1992).

Antioxidative properties of xanthan on autoxidation of soybean oil

in cyclodextrin emulsion. Journal of Agricultural and Food Chem-

istry, 40, 945–948.

Yang, B., Kallio, H., Tahvonen, R., Kalimo, K., Mattila, L., Kallio,

S., et al. (2000). Effects of dietary supplementation of Seabuck-

thorn (Hippophae rhamnoides) oils on fatty acids in patients with

atopic dermatitis. Journal of Nutrition and Biochemistry, 11(6),

338–340.

Yen, G. C., & Duh, P. D. (1993). Antioxidantive properties of

methanolic extracts from peanut hulls. Journal of the American Oil

Chemist�s Society, 70, 383–386.